Maintaining normal glucose levels in vivo is a crucial way that diabetic patients can avoid the long term problems associated with diabetes such as retinopathy, circulatory problems and other sequelae. For this reason, diabetic patients regularly monitor their blood glucose levels to, for example, optimize insulin dosing. In this context, a variety systems and methods have been developed for monitoring blood glucose levels. One strategy detects glucose levels using fluorescent compounds, for example with a competitive binding assay where glucose and fluorophore labeled glucose ligands/analogs compete for the binding site of glucose receptors and the resulting change in fluorescence is translated to a glucose concentration.
In certain competitive glucose binding assays, dextran is used as a displaceable glucose ligand. In such assays, dextran can be labeled with a lipophilic (and cationic) dyes such as hexamethoxy crystal violet-1 (HMCV1). However, the presence of a large number (e.g. greater than 10) of lipophilic dye molecules coupled to the flexible poly-(1,6)-glucose backbone of dextran can cause such labeled dextran molecules to adopt less water soluble conformations, which can result in the precipitation of these molecules. Moreover, such dye-induced conformal changes can drive the labeled dextran into a more lipophilic state, which can cause adverse changes on the binding ability of dextran to the glucose receptor as well as affecting its Förster Resonance Energy Transfer (FRET) efficiency. In addition, in conventional systems, dyes can be shielded intra-molecularly on the dextran, a phenomenon which can result in the calibration of the assay changing over time, thereby introducing instability into the assay.
Reference dyes are also used in certain fluorescence assays in order to track of variations in the experimental setup, e.g. light source fluctuations, changes in the optical path (coupling light into light guides, mechanical perturbations like bending, temperature variations, etc.). Traditionally, optical or fluorescence-based sensing systems have utilized reference fluorophores that are red-shifted relative to the assaying fluorophore. However, by exciting fluorophores using light of a lower wavelength with more energy than needed, there is an increased risk that the electronic transition in the fluorescent molecule will occur from the electronic ground state (S0) to the second excited state (S2) and not to the first excited electronic state (S1). Molecules in S2 are much more likely to decompose than the same molecule in S1. Hence, faster photobleaching occurs if the fluorophores are excited to S2 instead of only S1.
Accordingly, there is a need in the art for optical glucose assays that utilize agents and materials selected to enhance assay stability. The invention disclosed herein meets this need for example, by using assays designed to include multi-labeled glucose analogs/ligands (e.g. dextran coupled/labelled with agents that promote hydrophilicity) and/or blue-shifted reference fluorophores. As discussed below, glucose assays designed to include the multi-labeled glucose analogs and/or blue-shifted reference fluorophores exhibit improvements in material properties such as assay stability.